B cll isolation kit

Healthy donor PBMCs or isolated NK cells were stimulated overnight with IL-15 (1ng/ml; R&D Systems) and co-incubated with target cells at 10:1, 5:1 or 1:1 effector:target ratios as indicated for 4 hours with anti-CD107a-AF647. For assays using primary CLL cells as targets, CLL cells were isolated using a B-CLL isolation kit (Miltenyi Biotech) before co-incubation with healthy donor PBMC. For assays in combination with anti-CD20 antibodies, target cells were incubated with indicated antibody (1μg/ml; in-house) for 20 minutes at 4°C prior to addition of PBMC or purified NK cells (Miltenyi Biotech NK isolation kit) at a 10:1 effector:target ratio. Excess anti-CD20 antibody in the medium was removed by two washes with RPMI media prior to addition of PBMC. Golgistop (BD Biosciences) was added 1 hour after co-incubation and cells were then stained with REA147-FITC (Miltenyi Biotech), CH-L-PE (BD Biosciences), CD56-PE/Cy7 (Biolegend) CD3-PerCP (Biolegend) and analysed by flow cytometry on a BD FACS Aria. Data was analysed by FlowJo_V10 software.